Traditionally, calf rennet has been used as a milk coagulant in the production of cheese. In recent years, dramatic increases in worldwide cheese production and decreases in calf rennet supplies have stimulated the use of alternative milk coagulating enzymes. Among the available enzymes suitable for this purpose, the microbial rennets are favored because they can be mass produced and offer a variety of properties permitting selection of those most suitable for cheese production. A microbial rennet which has achieved significant commercial success is obtained during the fermentation of an organism from the species Mucor miehei. In the traditional method of preparing microbial rennet from this organism, the fermentation is carried out in a suitable nutrient medium and purification is carried out by filtering the fermentation broth, adjusting the pH of the filtrate, concentrating the filtrate and then using ultrafiltration and vacuum evaporation techniques to complete the purification. While this method is reasonably effective, carbohydrates, pigments and extraneous enzymes tend to accumulate in the final product. Furthermore, the targeted recovery of 85% enzyme activity is not always achieved.
Kobayashi, et al report in Analytical Biochemistry, 122, 308-312 (1982) that microbial rennet from M. miehei and Endothia parasitica was purified by the use of affinity columns including N-acetylpepstatin affinity gel.
Subramanian reports in CRC Critical Reviews in Biochemistry, Vol. 16, Issue 2, pages 169-205 (1984) that Cibacron Blue F3GA(CB) has ascended in status from a lowly textile dye to a glorified "biospecific" ligand in less than a decade.
Dean, et al in J. Chromatogr., 165, 301 (1979) and Burgett, et al in Am. Lab., p. 74 (1977) enumerate a large number of enzymes which have successfully been separated on a CB column. These enzymes include hexokinase, DNA-polymerase, alcohol dehydrogenase, adenylate kinase, ribonuclease, glyoxalase 1, cytochrome C, aldolase, blood clotting factor X and enolase. The enzymes do not have many features in common either in terms of structure or function.